There is a continuing and even more urgent need for a simple method and means for directly detecting the real or potential activity of chemical substances as carcinogens, including teratogens which are responsible for birth defects. Every year, thousands of new chemicals are developed which require screening for possible carcinogenic or mutagenic activity before approval for their intended purpose. Moreover, more sophisticated scrutiny of presently known and used chemicals initially considered to be benign, gives rise to increasing suspicion as to the hazards presented by their continued utilization calling for fresh evaluation of their acceptability. Further, as the protection of the work force is given increasing national priority, more efficient and more practical ways for determining the existence in the work environment of hazardous chemicals becomes a virtual necessity.
Considerable attention has already been devoted to this need, and a plethora of patents has been issued directed to various of its aspects. In one general category fall badge or strip type monitoring elements, often referred to as dosimeters. The following are a few U.S. patents that are representative of this group:
U.S. Pat. No. 3,693,327--Carbon monoxide is absorbed by a filter comprising hemoglobin which upon saturation undergoes a characteristic change in red coloration;
U.S. Pat. No. 4,205,043--Toxic gases, e.g., in a fire atmosphere, are detected by discs of absorbent material carried on a plastic substrate and covered by a peelable protective film, the discs containing a color-sensitive indicator for reacting with the toxic gas to distinctive color change;
U.S. Pat. No. 4,258,000--A microporous transparent polymeric matrix has its pores filled with a mixture of a solvent for the toxic agent to be detected and a color indicating reactant for reaction with the thus absorbed toxic agent;
U.S. Pat. No. 4,341,642--A partially hydrogenated sequenced styrene-butadiene copolymer undergoes rapid deterioration upon contact with certain organic pollutants, particularly hydrocarbons including halogenated hydrocarbons and oxygenated solvents, the deterioration triggering an indicating signal;
U.S. Pat. No. 4,421,719--A colorimetric indicator sensitive to the presence of a hazardous substance is adhered to a film backing with a high surface area carrier, e.g., alumina or silica, and a self-adhering bentonite clay mineral binder, the selection of the indicator depending on the particular hazardous substance to be detected, such as a permanganate salt for reactive detection of carbon monoxide.
U.S. Pat. No. 4,597,942--Ethylene oxide is detected as a suspect carcinogen by means of a cholesteric liquid crystal composition held in a binder to a substrate, such composition undergoing a characteristic color change when exposed to ethylene oxide; and
U.S. Pat. No. 4,680,165--A porous web is impregnated with an agent such as a heavy metal compound, for promoting the absorption and enhancing the phosphorescence of the vapors or aerosols of certain organic chemicals, especially polynuclear aromatic compounds, the absorbed material after a given period being examined for room temperature phosphorescence indicative of the amount absorbed.
Detection elements within this category have the important virtues of light-weight, portability and a generally direct acting response, but their sensitivity is limited to noxious substances for which there is a known companion colorimetric indicator. However, such colorimetric indicators tend to be specific to certain substances to be detected or at least to limited classes of such substances, and the concurrent detection of a broad spectrum of noxious substances in this fashion becomes complicated if not practically impossible.
In an effort to overcome this serious deficiency, researchers in the field have resorted to biological mechanisms to develop what are generally known as "bioassays," one of the most widely used and successful of such assays being commonly referred to as the "Ames test."
The literature is replete with papers relating to the Ames test; a review by Ames appears together with an extensive bibliography as of 1983 in Cancer, Volume 53, pages 2034-2040, May 25, 1984. to which reference may be had for more complete information. The Ames test is based on the principle that the mutational tendency of certain strains of bacteria will be altered by exposure to a carcinogenic or mutagenic substance. Specifically, the test employs a mutated strain of Salmonella typhimurium which lacks the ability to produce the essential amino acid histidine and consequently is incapable of multiplying when cultured in a nutrient medium lacking this essential nutrient. However, this mutated strain tends to revert to its wild or natural state in which it is able to produce histidine and thus grow when cultured, and this reversionary tendency is significantly increased by contact with a carcinogenic or mutagenic substance. Thus, when the mutated bacterial strain is cultured in contact with the substance to be tested, the reversionary mutagenic effect on the substance can be determined by counting the growth of the test plates and comparing the results against control plates indicative of the spontaneous reversionary tendencies of these bacteria in the absence of the test substance.
The human system, and indeed that of mammals generally, is endowed with many pathways for carrying out the metabolic oxidation of chemical substances into metabolic intermediates which may or may not then be disposed of or possibly subjected to additional pathways of oxidation. These pathways are referred to collectively as "mixed function oxidation" activity and are performed primarily by cells present in the liver and in other organs. According to P. J. O'Brien (Jun. 30, 1988) "Free-Radical-Mediated Chemical Carcinogenesis," Annals of the New York Academy of Sciences, volume 534, pp. 552-564, the metabolic intermediates so generated include various combinations of free radicals. They also include epoxides, aldehydes, sulfoxides, hydroxylammonium derivatives, organic aminohydrins and halohydrins, and the like, depending on the parent compound being oxidized. According to D. W. Nebert and F. J. Gonzalez (1987) "P450 Genes, Structure, Function, Evolution and Regulation," Annual Review of Biochemistry, Volume 56, pp. 945-993, these oxidations are carried out by at least eight families of membrane-bound enzymes containing cytochromes P-450. Recognizing that it is these ultimate metabolic intermediates that are actually systemically effective rather than the original or starting substance, the Ames test incorporates a metabolically active biological component exhibiting mixed function oxidation activity so as to approximate the multiple natural oxidative pathways occurring within the body so that the response of the bacterial strain is due to the metabolic intermediates generated by this component rather than to the original substance.
The Ames test incorporating the metabolically active biological component having mixed function oxidative activity has been carefully scrutinized and has been found to provide a remarkably good indication of the carcinogenic or mutagenic activity of a broad spectrum of chemical substances and, as of 1983, was said to be in use in thousands of laboratories for this purpose. Its sensitivity is by no means perfect, inasmuch as it is nonresponsive to some substances of high recognized toxicity, notably heavy metal compounds, but it certainly represents an important stride forward in the biochemical testing field in expanding the scope of application to a highly useful wide range of different test substances as contrasted with the specificity of dosimeter type test techniques.
Variations and improvements have been made on the Ames test in order to increase its usefulness. In U.S. Pat. No. 4,256,832, instead of visually counting the incubation results of the test plates, the incubation is rather carried out in a controlled oxygen-containing atmosphere from which the consumption of oxygen by the bacteria is detected and taken as a measure of the relative growth of the mutated strains.
In U.S. Pat. No. 4,299,915, the backward acting approach of the Ames test, dependent on the reversionary tendency of a mutated bacterial strain, is replaced by a forward acting assay which determines the effect upon the mutational capability of a different bacterial strain by the presence of the test substance. Here, a strain of S. typhimurium naturally possess the ability to enzymatically convert a purine analog, such as 8-azaguanine, into a toxic metabolite which thus inhibits the growth of the bacteria; whereas a mutant of the bacteria lacks the enzymes active on the purine analog, allowing the mutant cells to grow uninhibited when plated, and the mutational change is greater in the presence of a carcinogen or mutagen. Here again, use is made of a metabolically active component similar to that of the Ames test in order to convert the test substance to its ultimate metabolic state.
A somewhat different approach is taken in U.S. Pat. No. 4,072,572 which is based on the capacity of carcinogens or mutagens to decrease the fidelity of in vitro DNA synthesis by DNA polymerases from synthetic polynucleotides incorporating separately labelled correct, complementary nucleotide and incorrect, non-complementary nucleotide. The frequency with which the incorrect nucleotide is incorporated compared to that for the correct nucleotide, represents an "error frequency" indicative of the carcinogenic or mutagenic activity of the test substance present. Again, this procedure envisions the provision of metabolically active biological component having mixed function oxidase activity as in the Ames test for the same reason.
A less related bioassay is covered in U.S. Pat. No. 4,264,729 which presumes that a carcinogenic substance will preferentially promote the growth of cells that are already in a cancerous state compared to healthy tissue. Because the so-called carcinogenic activity of a given test substance tends to be specific for certain types of cancer, the procedure must employ pairs of diseased and healthy tissue from several different sites or organs of the body which are prone to develop cancer, e.g., the lung, breast, ovary, and kidney. This assay is capable of differentiating not only carcinogenic and toxic substances from neutral substances, but also of identifying substances which are specifically anti-cancerous or inhibitive of carcinogenic activity.
It is also known to base bioassays on immunogenic phenomena, one example being U.S. Pat. No. 3,903,898 wherein antibodies against carcinogenic antigenic agents from tobacco smoke are induced in test animals and the resultant antibodies are collected and employed as a filter for absorbing the antigenic agents in tobacco smoke by means of an immunological complexing reaction therewith.
Finally, it is known in U.S. Pat. No. 4,506,752 to utilize a dye in chemically reduced or leuco form, such as the leuco form of methylene blue or triphenylmethane dyes, as a colorimetric indicator for the adventitious entry of oxygen into a sealed oxygen-free package or the like.
Obviously, the Ames test and other bioassays described above must be carried out by skilled personnel and, at best, require a considerable period of time to develop useful results. Consequently, these tests are of limited applicability, notwithstanding the range of substances within their scope.